Towards a systematic map of the functional role of protein phosphorylation

Abstract

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphorylation sites have a known function. Here, we devised a chemical genetic approach to study the functional relevance of phosphorylation in S. cerevisiae . We generated 474 phospho-deficient mutants that, along with the gene deletion library, were screened for fitness in 102 conditions. Of these, 42% exhibited growth phenotypes, suggesting these phosphosites are likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. While some phospho-mutants showed loss-of-function phenotypes, a higher fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This study provides a roadmap for functionally characterizing phosphorylation in a systematic manner.

Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome

“…Thermal proteome profiling (TPP) is an emerging method that is revolutionizing how MS is able to gain insights into the proteome. TPP has been used to study such events as target engagement, posttranslational modifications, and cell cycle variation (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)76), however it has not yet been applied to biological questions regarding changes that occur as a consequence of single-gene missense mutations known to cause defects in cellular fitness through unknown mechanisms. We have applied TPP (Figure 2A) (57,77) to address the global effects of TS-inducing missense mutations on the proteome which we refer to as mutant TPP (mTPP).…”

“…Prior TPP experiments have revealed both direct and indirect consequences of on-and off-target drug and ligand binding through shifts in protein melt, suggesting that it could have broad applicability to discover biophysical changes in proteins in varying conditions (41)(42)(43)(44)(45). The principle that changes in protein context can be implied from alterations in thermal stability has also been used to study protein binding metabolites or nucleic acids (43,(46)(47)(48)(49), posttranslational modifications (50)(51)(52) or changes in redox status (53). Additionally, TPP has been used to analyze protein complex behavior in steadystate conditions (40) and throughout different stages of the cell cycle (46,54).…”

Thermal proteome profiling for interrogating protein interactions

“…In theory, any cell type can be used, provided that the lysis method does not resolubilize the heat‐induced insoluble protein fraction. To date, the method has been used to profile bacteria (Peng et al , ; Mateus et al , ), yeast (Ochoa et al , ; preprint: Viéitez et al , ), intracellular parasites (Dziekan et al , ), plant cells (Volkening et al , ), or mammalian cells (Savitski et al , ).…”

“…When using dose‐ or time‐dependent perturbations, samples from a single temperature can be combined in the same mass spectrometry run—an approach termed TPP compound concentration range (TPP‐CCR; Savitski et al , ; Franken et al , ), or if multiple temperatures are analyzed sequentially, two‐dimensional TPP (2D‐TPP; Becher et al , ; Fig ). Recently, the 2D‐TPP approach has been extended to discrete perturbations to study the human cell cycle (Becher et al , ; Dai et al , ), the effect of gene knock‐outs (Mateus et al , ; Banzhaf et al , ), or point mutations (Ochoa et al , ; preprint: Peck Justice et al , ; preprint: Viéitez et al , ). In the 2D‐TPP approach, melting curves for each protein cannot be obtained, since the lowest temperature sample (the reference sample for calculating the remaining soluble fraction at each temperature) is not present in all samples.…”

Recent advances in isobaric labeling and applications in quantitative proteomics

“…Since TPP's inception, there have been several new methods developed to improve and expand its applicability [ 131 ]. TPP has been expanded to monitor membrane proteins, phosphorylated proteins and peptides via phosphoproteomics combined with TPP (phospho‐TPP), bacteria, plants, plasmodium, yeast, viruses, tissue samples, and plasma membrane proteins [ 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 ]. Other protocol‐based methods include a two‐dimensional approach that studies protein abundance and regulation changes simultaneously, a proteome integral solubility alteration (PISA) to increase throughput and reduction in data analysis, and utilization of a vacuum manifold to increase throughput [ 146 , 147 ].…”